Inoculation Studies with the Wilt Fungus Verticillium Albo-Atrum

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INOCULATION STUDIES WITh Ti-lE WILT FUNGUS VERTICILLtU.: ALBO-ATRUM

LIANG TUNG LIN

A THESIS

submitted to the

OREGON STATE AGRICULTURAL COLLEGE

in partial fulfillment of the requirements for the degree of

MASTER OF SCIEIICE

June 1937 Redacted for privacy

Professor of Plant Patholoy In Charge of Major Redacted for privacy

Chairnan of School OEraduate Committee Redacted for privacy -______Chairman of Co11eg' Graduate Council

I ACIOWLEDGEMEN T S

I wish to express my sincere appreciation to Dr. C.

E. Owens, Head of the Department of 3otany and Plant

Pathology, for suggesting this investigation and his constant advice and willing assistance in carrying out the experimental work and preparing the thesis, and to

Dr. D. P. Rogers, Dr. S. M. Zeller, and other members of the staff of both the Department of Botany and Plant Path-. ology and the United States Department of Agriculture at

Corvallis, Oregon, for their helpful advice. TABLE OF CONTENTS

Introduction

Review of Literature

The Present Investigation

Plan of the Work

Source of Inoculum

Kinds of Plants Inoculated

Methods of Investigation

Inoculation

On Peach, Cherry, Prune

On Tomato

Checking Results

Symptoms Manifested

Re i s ola t i on s

Eistological Examination

Discuss ion

Summary

Literature Cited

Illustrations ILOCULATION STUDIES WIT1-i THE WILT FUNGUS VERTICILLItJM ALO-ATRtJM

Introduction

Verticilliuni wilt disease especially caused by the species Verticillium albo-atrum Reinke and Berthold is

widespread in tìe United States and abroad (25, 28, 29). Since the fungus, Verticillium albo-atrum, was described and named by Reinke and Berthold in 1879 in Germany as the cause of a wilt disease of potato, the attention of inves- tiators in Europe and America has been called to its eco- noclic importance as a pathogene of economic plants. It has been found that the fungus and its related secies attack a large number of plants. Over one hundred and twenty species included in tMrty-five widely unrelated families and eighteen orders (28, 29) have been reported as susceptible to attacks of this fungus. The list includes annual and ocrennial, herbaceous and woody plants. The common hosts of Verticillium albo-atrurn R. and 13. include potato (8, 9, 12, 20, 21, 23, 25, 28, 29), tomato (4, 5,

27, 28, 29), eggplant , 8, 9, 16, 20, 28, 29, 34), snapdragon (5, 7, 8, 9, 20, 28), raspberry (1, 2, 9, 19, 28), apricot (11, 28), maple (10, 14, 26, 28, 30), cnerry (28, 29, 33), plum, prune (28), etc. In western Oregon, Verticillium albo-atrum is more important than Fusrium oxysporuin as a cause of' potato wilt (21, 29, 23), and also as the cause of' wilt on cane fruits (33)..

In order to demonstrate experimentally the ability of Verticilliurn albo-atrum R. and B. to produce wilt disease of different plants and to gain a knowledge of the behavior of the fungus isolated from various sources 'zhen inoculated into other host 2lants, therefore, the investigation reported in this paper has been carried out. One of' the oects of this work has been to determine whether the isolates of Verticilliurn secured from various herbaceous and woody host plants in Oregon will readily attack such tree hosts as the peach, cilerry and prune.

Review of Literature

The general history and literature concerning the

Verticillium disease in various plants have been mentioned more completely by Rudolph (28) and Van der Meer (29).

Verticillium aibo-atruni possesses a wide host range as mentioned above, so its diagnostic characteristics may be somewhat variable depending upon the various hosts and environmental factors. Comparing the descriptions of the symptoms caused by this organism as given in different papers (25, 14, 11, 22, 4, 5, 15, 1, 24, 29), they may be summed up in a general way and the variations in symptoms also may be pointed out as follows: The infection y the fungus takes place on the under- ground parts of the host. The mycelium of the fungus pene- trates either the wounded or intact roots, passes into the wood, and grows up gradually through the vascular bundles into the stem, so that it causes abnormal phenomena showing on the aDove ground parts of the host, as a yellowing and wilting or the leaves. Such being the case, the disease first becomes apparent by a premature yellowing and drop- ping of the lower leaves of infected plants. This process advances upward, until the whole plant dies. Leaves, sometimes or quite often, become yellow along one side of the stem, or in other words the longitudinal halves of the stem are not subject to the infection in the same degree. Plants attached by tnis fungus usually are somewhat stunted. This is due to the fact that the internodes, especially the younger, are poorly developed. On woody plants it may cause the death of twigs and branches of the infected plant (15, 29). Gravatt (14) reported, in the case of riaple wilt, that sotie limes or parts of trees are idiled during the dormant season and do not leaf out in the spring. Bewley (5) reported on tomato that yellow biotcnes appear on 4

individual leaflets on tile lower leaves and these leaflets

wither, while in California yellow blotches may or may not

occur on the leaves of infected plants (28). Orton (24)

and 3ewley both have found out, on potato infected with

wilt, as well as on tomato, that when the diseased plant

is dead. the fungus wIthin it grows out to the air and pro-

duces spores on the sta.L near the ground.

On cutting open a diseased plant in a longitudinal direction, it will snow that the wood is distinctly brown

in color, and the xylem vessels are filled with myceliurn which can be seen with the microscope. In woody plants, darkening of the wood portion of aU. affected characteristic. Browning of the woody portion Df herbaceous stems also occurs. This wood. discoloration can generaliy be traced from roots up to the stem. Van der

Meer (29) reports the wood discoloration in the diseased cherry tree as showing a brown wood cylinder, surrounded. by a narrov; uncolored border, or partly reaching the cam- bium, and in branches, it often developes more strongly on one side, with a frayed edge.

The general wilting of the diseased plant may be recognized in late stages (33), but sometimes it appears suddenly (14). Bewley (1), in reporting on the sleepy disease of the tomato, stated that wnen the conditions of 5

temperature and liGht are favorable to the oran1sm, the disease symptoms appear quite suddenly and the plants wilt while still green. ie also said, in his next report (5), that the process of death is much slower under conditions less favorable to the causal organisai. This is similar to the report on potato wilt by Orton (25). He explained tiiat t'ne fact that it caused the death of the infected plants slo;';ly in Germany is possibly attributab1c to tern- perature and rainfall factors. McKay (22) has explained the phenomena of the wilting of the infected plant as follows: The growth of the fungus in the tissues of the stern and roots results in a plugging of the water-con- ductin vessels, the production of poisonous substances, or the destruction of the tissues to such an extent that normal functions cannot be performed. Under such circuril- stances the leaves lose their turgidity and become yellow and wilt. The whole plant finally dies. However, in sunny weather the infected plants may wither rapidly. During the night they often recover their turgidity for the time being. Regarding the success or failure in obtaining infection in cross or direct inoculation experiments by the former investigators the following citations from the literature may be mentioned. Successful inoculations. In 1914 and 1918, Carpenter

(8, 9) reported successfully to have inoculated okra with

Verticilliwn albo-atrum isolated from okra, potato, egg-

plant and snapdragon; also eggplant with the fungus from

okra, snapdragon and eggplant; and cotton with Verticilliurn

from okra.

In 1913, Jagger and Stewart (20) succeeded in inocu-

lating barberry with Verticillium from eggplant and vice versa. They also infected maple with the fungus from barberry and eggplant, and eggplant with Verticillium from

otato.

In 1921, 1922 and 1923, Bewley (3, 4, 5) successfully

inoculated eggplant, Capsicum sp., cotton and sycamore maple with Verticillium from to.iato; and in 1928, he infected potato, tomato, cucumber, melon, sweet pea, snapdragon and elm with Verticilliuin albo-atrum from any one of these same hosts (6).

In 1923, Weimer (31) also successfully inoculated eggplant with Verticillium albo-atrum from udo.

In 1924 and 1926, Haenseler (17, 13) reported that he successfully infected eggplant with Verticillium albo-atrum from peach and maple.

In 1925, Van der Meer (29) reported the successful inoculation of' Aconitum napellus L. with Verticillium 7 albo-atrum from Solanum lycopersicum Tm. and cherry; and Lupinus polyphyllus Lindi. with the fungus from Solanurn lycopersicuxa Tm.., Cucuinis sativus L. and cherry. In the same paper (29) he also mentioned that Phlox decussata Hort. was infected with Verticil1i1.r:i albo-atrum from Solanu:u tuberosurn L., Solanuna lycoersicurn Tm., Cucumis L., and cherry; Solanurn tuberosurn was infected with the fungus from Solanum oper Tm., Cucumis sativus L., and cherry; 3olanum lycopersicu Tm. was infected '::ith the fungus from Solanurn tn.berosum L., Cucumis sativus L., and cherry; and Cucumis sativus L. was infected with tue fungus from Solanum tuberosuni L., Solarium lycopersicum and cherry. In 1927, .Vorma1d (32) reported successful inoculation of potatoes with 1lerticillium from hops. In 1931, Rudolph (28) reported successful inoculation of California poppy, bum clover, dandelion, and groundsel with Verticillium from raspberry; Senecio vulgaris L. with Verticillium from apricot; LledicaRo hispida Gaertn. with Verticillium from apricot and tornato; common mallow with Verticilliurn from toato and raspberry. He has also infected myrobalan seedlings with Verticillium from peach, apricot and. raspberry; and raspberry plants with Verticillium from apricot, peach and tomato. In 1936, ¿eller (33) reported successful inoculation

of Oregon grown black raspberrios--Cumberland, Munger, and

Plum Farmer, v;ith Verticifl lho-atrm from black rasp-

berry canes and potato tubers.

Unsuccessful inoculations. In 1918, Jagger and

Stev:art (20) failed to infect barberry and eggplant with

Verticillium from maple, or maple with Verticillium from

potto..

In 1922, Haenseler (16) did not succeed in infecting

pepper and tomato plants v,'ith Verticillium from eggplant.

In 1924, Haenseler (17) was unsuccessful in infecting

peach seedlings and twigs with Verticilliuin from eggplant,

okra or peach; he also failed to produce the disease in

healthy peach twigs by budding them with buds from slightly

diseased peach twigs.

In 1925, Van der Meer (29) did not succeed in infect-

Ing Aconi nape11us L. with V. albo-atrum from Solanum tuberos'm L. or Cucumis sativus L.

Present Investigation

Plan 2.: work. In these investigations it was planned to collect a number of cultures of Verticillium albo-atrum from a number of different species of host plants and to carry out inoculation experiments in the greenhouse in an attern)t to determine the cross inoculability g

of various strains upon a number of different hosts, parti-

cularly including the stone fruits, peach, cherry and prune.

It has been generally known for a long time that such plants

as potatoes, tomatoes, eggplants and raspberries vrere

apparently cross inoculable by a common strain of Verticil-

1juin prevalent in the Willamette Valley and other sections

of Oregon. Verticillium wilt has also been found in orna- mental maple trees in Oregon. To what extent the stone

fruit trees such as peach, cherry and prune may be subject

to the prevalent strain al' Verticillium in Oregon was not known. Neither was it iiovm whether the Verticillium strain

on maple is identical with the common strain of Verticillium

nor whether it would attack the stone fruit trees as v;ell

as maple.

Therefore the following plan of investigation was

csrried out.

Source of inoculum. Five strains or isolates oÍ' Verti-

cillium were used in these investigations as follows:

Isolate !:o. E 920 from maple.

Isolate No. E 923 from tomato.

Isolate No. E 925 from eggplant.

Isolate No. E 926 from snapdragon.

Isolate o. E 933 from raspberry.

Plants inoculated. Sii ice tomato plants are easily

grown in the greenhouse and are uite susceptible to lo

Verticillium wilt they were used as a check to determine the relative virulence of the various isolates or strains. Three representatives of the stone fruit group, peach, cherry and prune, viere used in an attempt to determine if any of the five isolates would infect them.

Method of' Investigation

In order to find out the ability of Verticillium albo- atrum Reinke and Berthold to produce wilt disease of different plants and to gain a knowledge of the relation of the fungus isolated from various sources to other host lants, therefore, inoculations viere performed on certain plants with different inocula which were isolated from maple, tomato, eggplant, snapdragon and raspberry, as mentioned above. Plants---tomato, peach, cherry and prune, used for inoculating in the experiments were planted in pots and kept in the greenhouse. Peach, cherry, and pxine plants were all about two years old when the experimental work started. Inoculation. Inoculations were made by digging out the soil, breaking the small roots of the plant, and placing the inoculum in the soil in contact with the wounded roots in order to permit tue fungus to penetrate 11

easily. The inoculum must be covered with soil. The dif-

ferent strains of Verticillium albo-atrum isolated from

various sources were cultivated on sweet clover ste::s or

potato agar and used as inocula. Sometimes the soil used

for growing the inoculated tomato plant was sterilized in

tue autoclave before planting to keep out the soil contam-

inations in order that nothing may interfere with the

inoculation. Also, the tomato seeds :ere sometimes sown

directly on the sick soil which had been inoculated arti-

ficially in the prior crop, to see if the fungus would penetrate through the intact roots.

Inoculations on Peach, Cherry and Prune

Inoculations were made at various times on these potted

trees as indicated below.

Experiment No. 1.

On September 22, 1936, the Verticillium alho-atru.m

No. E 920 which was isolated from maple and cultivated on

sweet clover stems, was inoculated on the roots of two

peach plants--Late Crawford and J. H. Hale, two cherries

--apo1eon and Eontmorency, and one prune--Italian.

Experiment No. 2.

On October 19, 1936, three groups of five each of peach, cherry and lrune plants were inoculated with the 12

isolate No. E 920 (maple). Inoculum used consisted of a

pure culture of the fungus on sweet clover sterns.

Experiment No. 3..

On January 21, 1937, three peaches--Early Crawford,

Muir and J. H. Hale, one cherry--Napoleon, and one prune

--rtalian, were inoculated with the isolate No. E 920

(maple); four peaches--Slappy, Early Crawford, J. H. Hale

and uir, three cherries-.--Larbert, iapoleon and Montmorency,

and one prune--Italian, were inoculated with the isolate

o. E 923 (tornato); two peaches--Halberta and Slappy,

three che rrie s--Napoleon, Lambert and Montmorency, and

one prune--rtalian, were inoculated with the isolate

No. 925 (eggplant); two peaches--Slappy and Early Crawford,

two cherries-L1ontorency and Lambert, and one prune--

Italian, were inoculated with the isolate No. E 926. The

results from these inoculations are shown in Table IV.

Inoculations on Tomato

Experiment i:o. 4.

On November 9, 1936, groups of five tomato seedlings

each were inoculated with the isolates Nos. E 920 (maple),

E 923 (tornato), E 925 (eggplant) and E 926 (snapdragon)

respectively; and five seedlings were left for chechs. The

inocula of these different strains were grown in pure cul-

ture on sweet clover stems. 13

Experiment ITo. 5.

On January 22, 1937, to:ato seeds which had been

sterilized with a i to 1000 solution of mercuric chloride

and rinsed thoroughly in sterile distilled water were

sown in )ots in which the soil had already been inoculated

in the following manner: These pots had previously grovin

a crop of tomatoes which had been inoculated and become

infected with Verticillium. These k;OtS containing sick

soil consisted of groups of five each inoculated vith the

isolates E 920, E 923, E 925, and E 926 respectively. Some

seeds were sown in a number of pots containing sterile

soil as checks. This planned to determine

whether infection could take place through the intact roots

of seedling tomatoes.

Experiment No. 6.

On March 26, 1937, groups of five each of tomato plants which had been grown in soil sterilized in the autoclave were inoculated with the following isolates respectively:

E 920 (male), E 923 (tornato), E 925 (eggplant), E 926

(snapdragon) and E 933 (raspberry). These were grown as a check against those grown in unsterilized soil in previous series. Five pots v:ere left uninoculated as checks. 14

Checking Results

After the experiments had run a sufficient length of

time, the results were checked in three ways, namely, (a)

by observing the symptoms manifested by the inoculated

slants as compared with the checks; (b) by attempting to

reisolate the fungus from inoculated as well as check plants;

and (c) by sectioning sterns and roots and e::arnining them microscopically for the presence of myceliuin of Verticillium

in the tracheae..

Symptoms Manifested

Q.a tomatoes The results of inoculations on tomatoes

appeared as a general yellowing of foliage of the inoculated

plants. In most cases yellow blotches also appeared on

the infected leaflets (Plate 14). The yellowing spread

out and upwards and finally resulted in the foliage of the whole plant becoming entirely yellow (Plate 12). Leaves

sometimes became yellow unilaterally along one side of the stem (Plate 13). The fungus, Verticillium albo-atruin, from

different sources-maple, tornato, eggplant, snapdragon

and raspberry--attacked tomato Plants and while they did

not produce typical wilt symptoms under greenhouse condi-

tions the yellowing was conspicuous. Tomato plants inocu-

lated with the strain of Verticillium albo-atrurn from 15 maple, alvays siowed more serious symptoms. Leaves withered but some parts remained green. Sometimes it caused the infected ilant to be markedly stunted (Plate 11). There are also certain internal symptoms which may be very noticeable at times. In a longitudinal section through the stem and root of a diseased tomato plant, the brown stripes can be seen by the unaided eyes in the vascular tissue, and may be traced from root to top, if they are conspicuous (Plate 15).

On stone fruit trees. In Experirients o. 1, 2 and 3 most of the inoculated cherry, peach, and prune trees did not leaf out nrmal1y in the spring.. However, this condition was not confined. to the inoculated 1ants but occurred in the uninoculated plants also, therefore, it seems probable that this condition is due to causes other than infection by Vertici11iiu. In view of this failure of positive outward symptoms it was necessary to rely upon internal symptoms, reisolation trials and microscopic examination of sections of stems and roots for determination of the success or failure of infection in these trees.

Re i solations

From tomato. Vhen the symptoms of the disease liad already shown quite clearly an the inoculated plant, it 16

was dug out from the soll, washed thoroughly with clean

water and attempts to reisolate the fungus were :ade. Some

pieces of the plant tissue were cut out through the wood

on certain portions of the plant from the base to top,

soaked one rìinute in a i to 1000 solution of ::iercuric

chloride, rinsed through three changes of sterile distilled water and cultured on tue poured Petri dish plates of po-

tato glucose agar for the possible presence of the fungus,

Verticilliu;ri albo-atrum, in different portions of the p1ants

On January 12, 1937, attempts t reisolation from the inoculated and check tomato plants in Experiment No. 3 were made.. Of the five plants inoculated with each of the four different isolates positive results were obtained in the following degree: Of the five plants inoculated with isolate No. E 920, 5 became infected for a total 0±' lOO; of those inoculated with E 923, 5 gave positive results, or lOO infection; E 925 gave 2 positive or 4O; and E 926 gave i positive or 2O. The 5 check plants were all healthy.

According to this experiment the isolate from snapdragon

(E 926) attacks tornato less vigorously than the iso1ats from maple (E 920), tomato (E 923), or eggplant (E 925),

(Table I).

On May 7, 1937, the tomato plants grown in sick soil and sterile soil from Experiment No. 5 ;ere cultured in an 17 attempt to reisolate Verticil1iiirn. Those inoculated with

E 920 gave 5 positive out of 5, or 100%; E 923 gave 4 out of 5 positive, 80%; E 925 gave i out of 5 for a per- centage of 20; and E 926 gave none. Those grown in sterile soil also gave no positive results (Table II). The results from this experiment show that the Verticillium fungus can exist in the soil for some time and that infection can occur through the intact roots (Table II).

On May 17, 1937, the inoculated and uninoculated tomato plants from Experiment No. 6 were cultured. Five plants inoculated with the isolate No. E 920 (maple), the same number of plants inoculated with the isolate No. E 923

(tomato), three inoculated with the isolate No. F 925

(eggplant), and one inoculated with the isolate floe E 926

(snapdragon) gave Verticillium albo-atrum in culture.

The entire five plants inoculated with the isolate No.

E 933 (raspberry) and those uninoculated both gave no growth of Verticillium. In three of the five plants which were inoculated with the isolate No. E 920 (maple), two with the No. E 923 (tomato) and one with the No. E 925

(eggplant) the fungus could be reisolated from the upper portions of tops as weil as from roots and base of stm (Table III).

From the stone fruits. At intervals between May 19 and June 14, 1937, various samples of the inoculated stone 18 fruit trees, cherry, peach and prune, were removed from the pots and attempts t'ere made to reisolate Verticillium from the roots and base of stems. All attempts t reisola- ting the fungus gave neg;ive results. All tissue plantings on asar proved to be entirely sterile (Table IV).

Examination of Sections

Tomato. It was comparatively easy to find the mycelium of Verticillium in the xylem vessels of all tomato plants which showed by symptoms and by reisolation cultures that they were infected. On the other hand, the checks v:hich appeared healthy and which gave no fungus when cultured also showed the absence of mycelium in the vessels.

Stone fruit trees. From all of the cherry, peach and prune trees from which reisolations were attempted blocks of wood were taken for sectioning. The sections v;ere cut on a sliding microtome. Some of them were examined without staining while others were stained to ke evident any mycelium th:3t might be present in the xylem vessels. Io indication of the presence of mrceliurn was found in any of' the inoculated trees nor in any of the checks (Table IV).

Discussion

Experiments No. 4, 5, and ô demonstrate that tomato plants are susceptible to the different isolates of Verticilliurn used in this investigation but in varying de-

grees. Since these tests were very limited in extent and

carried out in the greenhouse it should be recognized that

there .ray be a possible experimental error in the.work, but

under the conditions prevailing and within the limits of

this investigation there seems to be some evidence that tomato plants are very susceptible to the strains from maple, tornato and eggplant; less susceptible to the strain

from snapdragon; and slightly or not at all susceptible to the strain from raspberry.

Evidence that the Verticilliun- fungus may survive in fallow soil for at least several weeks was brought out in Experiment No. 5 wherein tomatoes grown in pots were inoculated through the soil and allowed to grow for some weeks until thorouiily infected. Then these plants were removed from the pots and the soil in the pots was allowed to lie fallow for several weeks. Subsequently sterile tomato seeds were planted in this sick soil. The seeds produced plants which in due course became diseased without further inoculation. These plants yielded the Verti- cillium fungus in culture, and sections of the tissue showed mycelium of Verticillium in the tracheae.

The writer found that the myceliurn of the fungus within the host tissue nay be spread crosswise from one xylem vessel to another by way of the pits (Plates 16 and 17). 20

Different strains of Verticillium albo-atrum may be

distinguished in culture by the different degrees to Which

scierotla are fornd, as well as by the differences in

pathogenicity as :easured by intensity of symptoms and

the ease with which the fungus can be reisolated from

plants inoculated with the different isolates

Summary

1.. Different strains cf Verticilijum albo-atrum

isolated from maple, tomato, eggplant and snapdragon v;ere

able to attack tomato plants in varying degrees of intensity

as indicated by symptoms and by reisolation trials from

inoculated lants.

2. The inoculation trials upon peach, cherry and

prune were not conclusive. Under the unnatural conditions

in the greenhouse there was no clear cut difference in

outward symptoms between inoculated plants and checks.

Reisolation trials failed to recover the fungus, and mycelium was not seen within the tissues. The only apparent

symptom was the discoloration around the pith shown in

cross-sections of root and stem taken just below and just above the surface of the ground. Only a part of the inoculated plants showed this discoloration, but none of the checks showed such a symptom. Table I. Toiato plants inoculated on Iovember 9, 1937, (Experiment No. 4), with Verticillium cultures from different sources. Reisolation made on January 12, 1937.

No. of Reisolation of ource of inocu- Symptoms appeared Verticilliurn Inoculum lated on January 6, 1937 o. of Per plants ______Infections cent

aple 5 6 to 9 of 8 to 16 5 100% (E 920) lower leaves became yellow with blotches, leaflets mostly vaithered.

ornito 5 4to5ofl3tol9 ;3 ( 923) lower leaves die- colored, tips of leaflets mostly yellow and wither-- ed

Eggplant 5 1 to 4 of l2to 15 . 40% (E 925) lov,rer leaves discolored, leaflets mostly yellow and some withered.

riapdrago. 5 13 to 14 of 8 to 2( i 20 (E 926) ].ovier leaves be- canLe yellow, 1ef- lets mostly dry.

eck 5 Healthy 0% Table II. Tomato plants inoculated on January 22, 1937,

(xperimnent No 5), with Verticilliu: cultures

from different sources.

No.. of Reisolation of Source of inocu-. Symptoms appeared Verticillium Inoculum lated on March 26, 1937 No. of Per jants nfectiç sent

}Japle 5 5 to 7 of S to 10 5 ioo; (E 920) lower leaves became yellow, one plant stunted.

Tomato 5 5 to 7 of 7 to £ 4 10 (E 923) lower leaves became yellow, one plant stunted.

Eggplant 5 3 to 5 of 9 to 11 1 (E 925) lower leaves discolored and became yellow.

Snapdragon 5 3 to 5 of 9 to 12 0 (E 926) lower leaves discolored and became yellow.

Check 5 Healthy O o,: Table III. Tomato .dants inoculated on March 26, 1937,

with Verticilliuni cultures fror different

sources, (Experiment No.. 6).

-- ______I:o. of Reisolatfbn of $ouroe of inocu.- 3ymtoms appeared Vert.icillium Inoculum lated on April 24, 1937 To. of Per ______plants Infections cent

Maple 5 Leaves on lower 5 1OO (E 920) a1f mostly became yellow, some with blotches upwards near the top, some leaflets withered. 3 ______I)laflts stunted.

Tomato 5 Leaves nostly 5 lOO (E 923) with blotches, 2 plants leaves became yellow near the top, 2 D1ants - - - slightly stunted. Eggplant 5 Lower leaves 3 6O (E 925) mostly v;ith blotches, lowest one became yellow i plant yellow leaves just on One side.

Snapdragon b Lower leaves i 2O (E 926) ostly discolored and became yellow

Raspberry 5 Lower leaves O 0% (E 933) mostly with blotches and became yellow.

Check 5 Healthy O 0% Table IV. 'body plants (cherry, peach, prune) inoculated

with cultures of Verticillium from different sources.

Source of Inoculated Plants Plants Infected Inoculum Name No. To. Per cent

.:aple Cherry 3 0 0 (E 920) ______

Tomato Cherry 2 0 0 (E 923) ______

Eggplant Cherry 4 (E 925) ______

Snapdragon Cherry 2 0 0 (E 926) ______- -. Check ------_ Cherry O _____

Iviaple Peach 5 - (E 920) ______------

Tomato Peach 2 0 0 (E 923) ______- ______

Eggplant Peach 2 0 0 _(E 925) ______

Snapdragon Peach 2 0 0 (E 926) ______- Check -__Peach_ o o

Maple Prune 3 0 0 (E920) -

Prune i O O - (E923 ______

Eggplant Prune 1 0 0 (E925) ______-- Snapdragon Prune i O O (E 926) ______Jheck Prune - i O O Literature Cited

1. Berkeley, G. H. and Jackson, A. B. Verticil1iu "lit of the red raspberry. Sci. Agr. 6(8): 261-270. 9 fig. 1926.

2. Berkeley, G. H. and Jackson, A. B. Verticiliiu: Iwi1tt of the raspberry. Phytopath. 16(10): 755-756. 1926.

3. Bewley, W. F. "Sleepy disease" or vrilt of the tomato. Sixth Ann. Rept. (1920) Cheshunt Exp. and Res. Sta. p. 26-38. 1921.

4. Bewley, W. F. "Sleepy disease" of the tomato. Ann. Appi. Biol. 9(2): 116-134. 3 p1. 1922.

5. Bewley, W. F. "Sleepy disease" of the tomato. Jour. LIin. Agr. (Gr. Brit.) 30: 450-457. 2 fig. 1923.

6. Beweley, W. F. Diseases of glasshouse 9lants. 208 p. 47 fig. Ernest Benn, London. 1928.

7. Brown, N. A. A snapdragon wilt due to Verticillium. Phytopath. 4(3): 217. 1914.

8. Carpenter, C. V1. The Verticillium wilt problem. Phytopath. 4(6): 393. 1914.

9. Carpenter, C. W. Wilt diseases of okra and the Verti- cillium problem. Jour. Agr. Res. 12(9): 329-546. 12 1. 1918.

10. Colley, H. The maple wilt. Wisconsin Dept. Agr. Bui. 52: 68-78. 2 fig. 1922. li. Czarnecki, H. Studies on the so-called black heart disease of the appricot. Phytopath. 13(5): 216-224. 1 pi. 4 fig. 1923.

12. Edson, J. A. Vascular discoloration of Irish potato tubers. Jour. Agr. Res. 20(4): 277-294. 1920.

13. Edson, H. A. and Shapovalov, . Temperature relations of certain potato-rot and wilt-producing fungi. Jour. Agr. Res. 18(10): 511-524. 1920. 14. Gravatt, G. H. Maple wilt. U. S. Dept. Agr. Dept. Cir. 82: 1-13. 9 fig. 1926.

15. :-aense1er, C. L. A new peach wilt disease. Phytopath. 12(1): 56-5'?. 1922. 16. i-iaenseler, C. M. Studies on eggplant wilt. New Jersey Agr. Exp. Sta. 42 Ann. Rept. p. 469-470. 1922.

17. ilaenseler, C. M. Peach wilt. New Jersey Agr. Exp. Sta. Rept. Dept. Plant Path. 1922-23: 364- 366. 2 pl. 1924.

18. Haenseler, C. M. Maple wilt. (.:immeo.) New Jersey Agr. Exp. Sta.. Dept. Plant Path. Plant Disease Notes. 4(8): 2-3. 1926. 19. Harris, R. V. The blue stripe 'ut of the raspberry. Jour. Pomol. and Hort. Sci. 4(3 and 4): 221-229. 3 pl. 195. 20. Jagger, I. C., and Stewart, V. 3. Some Verticillium diseases. Phytopath. 8(1): 15-19. 1918.

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28. Rudolph, B. A. Verticilliuin hadromycosis. Hilgardia 5(9): 197-353. 4 pl. 1931. 29. Van der Meer, J. i. H. Verticiliiuii v;iit of herbaceous and woody plants. Eed. Landbouwiioogeschooi (Wageningen) 28: l-82. illus. 1925.

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34. Zimm, L. A. A wilt disease of maples. Phytopath. 8(2): 80-81. 1918. PLATE i

Different strains of Verticililurn albo-atrum on potato glucose agar, 18 days old, showing the different intensity in forming black resting mycolium and microscierotia in the cultures from different sources: (i) maple, (2) tornato, (3) eggplant, (4) snapdragon and (5) raspberry. p/I

1/ PLATE 2

Conidiophores, conidia, hyphae and

.:icrosclerotium of Verticilliuxri albo-atrum isolated from maple and cultured on potato glucose agar.

PLATE 3

Conidiophores and conidia of Verti- cilliurn albo-atrum isolated from tomato and cultured on potato glucose agar. O

j j

924 PLATE 4

Conid1opiores and conidia of Verti- cillium albo-atrum isolated from egplant and cultured on potato glucose agar. 0000 o 0c

PLATE 5

Cortidiophores and conidia of Verti- cillium albo-atrurn isolated from snapdragon and cultured on potato glucose agar. 9X6 PLATE 6

Conidiophores and conidia of Vert!- cilliurn albo-atrum isolated. from raspberry and cultured on potato glucose agar. 000

I.."

L 943 PLATE 7

Tomato plants: the right one healthy, the left one artificially infected with Vertici1l1vîj albo-atrun

(from maple) and showing the typical symptoms of Vertici1lhn wilt.

PLATE 8

Tomato plant8: the right one healthy, the left one artificially infected with Verticillium albo-atrum (from tomato) and showing the typical syraptoms of Verticl1ium wilt. Zb3 I PLATE 9

Tomato plant5: the right one healthy, the left one artificially infected with Verticilllum albo-atrum

(from eggplant) and showing the typical symptonis of Verticilliuni wilt. ç'-y! -1 PLATE 10

Tomato plant3: the right one healthy, the left one artificially infected with Vertiç1lium albo-atrum (from snapdragon) and showing the typical symptoms of Verticillium wilt.

PLATE 11

Tomato plants: the right one healthy, the left one showing the 2lant stunted and some leaves yellow and vithered, caused by artificial infection with Verticillium albo-trum (from maple). -. 4 ,.

.4.

ç '

..,.. .7_Iv PLATE 12

Tomato plants showing the progress of infection: the left one just showing the yellowing symptoms on lower part; the right one showing the infection advancing upward, the yellow blotches appearing on leaflets near the top of plant.

PLATE 13

Tomato plant artificially infected. with Verticillium albo-atrurn (from eggplant) showing the infection advanced upward at one side of the plant, leaves becoming yellow.

PLATE 14

Three tomato leaflets (left to right) 1. nonnal

leaflet, 2. with yellow blotches, 3. whole leaflet becoming yellow, showing the intensity of the disease caused by Verticillium albo-atrum.

PLATE 15

Two longitudinal sections through crown and root of a diseased tomato plant caused by Verticillium albo-atrum

(from tomato) showing browning of vascular tissues.

PLATE 16a

Photomicrograph of a part of a cross-

section of diseased tomato stern caused by

Verticillium albo-atrum (from maple) show-

ing the mycelium of the fungus grown within

the vessel and a hypha penetrating the cell wall and entering the next vessel.

PLATE 16b

Photomicrograph of longitudinal section of diseased tomato stern showing a hypha of the causal organism, Verticillium albo-atrum (from maple), penetrating through the pit of a vessel. I J 01 - j PLATE 17

A drawing oÍ' the same picture of Plate

16b showing more cler1y that the hyphae of the causal organism penetrate through the pits of vessels.

PLATE 18

Cross-sections of sterns arid main roots of 3-year- old woody plants inoculated with Verticilliurn albo-atrum (from maple) showing the browning in the region limne- diately surrounding the pith. (A) cherry stem, (B) cherry root, (C) peach stem, (D) peach root, (E) prune stem and (F) prune root.

The fungus could not be reisolated from these plants nor could myceliurn be found in sections of either the discolored area or other regions of the roots and stems. lxi